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Purity: ≥98%
NG 52 (NG-52), a tri-substituted purine, is a cell-permeable, reversible, and ATP-competitve inhibitor of the cell cycle-regulating kinase, Cdc28p with IC50 of 7 μM, and the related Pho85p kinase with IC50 of 2 μM. NG-52 inhibits Cdc28p and Pho85p by attaching to the ATP-binding site of yeast cyclin-dependent kinases.
| Targets |
cdc2-cyclin B (IC50 = 0.34 μM); Pho85p (IC50 = 2 nM); Cdc28p (IC50 = 7 μM); Phosphoglycerate kinase 1 (PGK1) (IC50 = 2.5 μM)
NG52 inhibits the kinase activity of phosphoglycerate kinase 1 (PGK1) with an IC₅₀ of 2.5 ± 0.2 μM, without affecting its phosphatase activity. Molecular modeling suggests it binds competitively with ATP at the PGK1 active site, forming three potential hydrogen bonds with Gly238 and Gly313. [2] |
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| ln Vitro |
NG 52 (Compound 52) stops drug-sensitized S cells from growing. strains of cerevisiae that have a 30 μM GI50. With an IC50 value of 340 nM, NG 52 has activity against cdc2-cyclin B[1]. While primary astrocyte proliferation is efficiently inhibited, NG 52 dose-regulatedly decreases the GI50 values of neuroastrocytoma U87 and U251 cell lines to 7.8 μM and 5.2 μM, respectively [2]. In U87 and U251 cells, NG 52 (12.5-50 μM) efficiently suppresses the phosphorylation of PDH at Ser293 and PDHK1 at Thr338. This increases the amount of pyruvate that enters the Krebs cycle, which in turn increases ATP and ROS. manufacturing[2]. By suppressing the activity of PGK1, NG 52 increases the activity of pyruvate dehydrogenase (PDH), which reverses the Warburg effect and shifts oxygen-assisted cells from anaerobic to aerobic mode [2].
NG52 dose-dependently inhibited the proliferation of glioma cell lines U87 and U251 with GI₅₀ values of 7.8 ± 1.1 μM and 5.2 ± 0.2 μM, respectively. It also potently inhibited the proliferation of primary glioma cells. [2] NG52 (12.5-50 μM) effectively inhibited the phosphorylation of PDHK1 at Thr338 and PDH at Ser293 in U87 and U251 cells under hypoxic conditions, leading to more pyruvate entering the TCA cycle, increased ATP and ROS production, and decreased lactate production, thereby reversing the Warburg effect. [2] NG52 treatment (various concentrations for 3 days) significantly increased Annexin V-positive cell populations in U87 and U251 cells, indicating induction of apoptosis. [2] NG52 downregulated the expression of EMT markers N-cadherin and vimentin in U87 and U251 cells. [2] Clonogenic assays showed that NG52 treatment for 6 days significantly reduced the number of colonies formed by U87 and U251 cells. [2] In U87 cells with PGK1 knockdown (sh#220), the antiproliferative effect of NG52 was diminished, supporting on-target inhibition of PGK1. [2] |
| ln Vivo |
Neuronal tumor xenograft growth is inhibited by dose-regulated NG 52 (50-150 mg/kg; basal; daily; for 13 days) [2].
In a patient-derived glioma xenograft (PDX) model in nude mice, oral administration of NG52 (50, 100, 150 mg·kg⁻¹·d⁻¹ for 13 days) dose-dependently suppressed tumor growth. Tumor growth inhibition reached 100% at the 150 mg/kg dose. No significant body weight loss was observed, indicating good tolerability. [2] |
| Enzyme Assay |
A high-throughput screening assay was used to identify PGK1 kinase inhibitors. Recombinant PGK1 was incubated with substrate 3-phosphoglycerate and ATP in a reaction buffer containing test compounds or DMSO in 384-well plates. After incubation, an ADP-Glo reagent was added to stop the reaction and deplete remaining ATP. A kinase detection reagent was then added to convert ADP back to ATP, which was quantified using a luciferase/luciferin system to measure luminescence. [2]
The inhibitory effect of NG52 on PGK1 phosphatase activity was tested by incubating purified PGK1 with the compound, then adding substrates (GAP, β-NAD, ADP, GAPDH). ATP production was detected using an ADP-Glo assay. NG52 did not inhibit phosphatase activity. [2] An ADP-Glo biochemical assay was used to determine the IC₅₀ of NG52 against PGK1 kinase activity. Recombinant PGK1 was incubated with substrate 3-phosphoglycerate and ATP in the presence of NG52 or DMSO. Detection followed the standard ADP-Glo protocol. [2] |
| Cell Assay |
Cell proliferation assay [2]
Cell Types: Glioma U87 and U251 Cell Tested Concentrations: 0 μM, 12.5 μM, 25 μM, 50 μM Incubation Duration: 6 days Experimental Results: Effectively inhibited the proliferation of primary glioma cells. Western Blot Analysis [2] Cell Types: glioma U87 and U251 cells Tested Concentrations: 0 μM, 12.5 μM, 25 μM, 50 μM Incubation Duration: 12 hrs (hours) or 24 hrs (hours) Experimental Results: Effectively inhibited the proliferation of primary glioma cells. For proliferation assays, glioma cells were seeded in 24-well plates and treated with various concentrations of NG52 for 6 days. Cell viability was assessed using a Cell Titer-Glo luminescent assay. [2] For colony formation assays, U87 and U251 cells were plated in 6-well plates and treated with NG52 or DMSO for 6 days. Colonies were fixed, stained with crystal violet, and counted. [2] Apoptosis was analyzed using an Annexin V-FITC/PI staining kit. Cells treated with NG52 for 3 days were stained and analyzed by flow cytometry. [2] ROS levels were detected by incubating cells treated with NG52 with DCFH-DA dye for 20 minutes, followed by flow cytometry analysis. [2] ATP levels were measured using a Cell Titer-Glo assay after treating cells with NG52 for 12 and 48 hours. Lactate levels were measured using a commercial lactate assay kit after 12 hours of treatment. [2] Western blot analysis was performed to detect protein expression (e.g., PGK1, p-PDHK1, p-PDH, N-cadherin, vimentin) in cells treated with or without NG52. [2] For hypoxia experiments, cells were cultured in an incubator with 2% oxygen concentration. Treatment and handling protocols were otherwise the same as under normoxic conditions. [2] |
| Animal Protocol |
Animal/Disease Models: Female nu/nu (nude) mice (5-week old) injected with glioma cells [2]
Doses: 50 mg/kg, 100 mg/kg, 150 mg/kg Route of Administration: oral; daily; continued for 13 Day Experimental Results: Dose-dependent inhibition of glioma xenograft growth. A patient-derived glioma xenograft (PDX) model was established in five-week-old female nu/nu mice. Primary glioma cells were injected intracranially. Two weeks after implantation, mice were randomized into treatment groups. NG52 or vehicle was administered daily by oral gavage at doses of 50, 100, or 150 mg·kg⁻¹·d⁻¹ for 13 days. Tumor growth and mouse body weights were monitored. [2] |
| Toxicity/Toxicokinetics |
In the in vivo PDX model, mice were given an oral dose of up to 150 mg·kg⁻¹·d⁻¹ of NG52 for 13 consecutive days without significant weight loss, indicating that NG52 has good safety at these doses in this model. [2]
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| References | |
| Additional Infomation |
NG52 was identified as a PGK1 kinase inhibitor through high-throughput screening. It was previously known to be a yeast cell cycle-regulated kinase inhibitor. [2] The mechanism of action of NG52 in glioma treatment is thought to be the inhibition of PGK1 kinase activity, thereby reversing the Wahlberg effect. This inhibition prevents PGK1-mediated phosphorylation of PDHK1 (Thr338), resulting in a decrease and inhibition of PDH (Ser293) phosphorylation. As a result, the flux of pyruvate into the tricarboxylic acid cycle is restored, oxidative phosphorylation, ATP and reactive oxygen species (ROS) production are increased, while glycolysis and lactate production are reduced. This metabolic shift inhibits glioma cell proliferation, induces apoptosis, and inhibits epithelial-mesenchymal transition (EMT). [2] This study suggests that targeting PGK1 kinase activity (rather than its glycolytic function) is a promising strategy for treating gliomas because it can specifically reverse the cancer-specific Wahlberg effect without affecting normal cell metabolism. [2]
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| Molecular Formula |
C16H19CLN6O
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|---|---|
| Molecular Weight |
346.81466
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| Exact Mass |
346.13
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| Elemental Analysis |
C, 55.41; H, 5.52; Cl, 10.22; N, 24.23; O, 4.61
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| CAS # |
212779-48-1
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| Related CAS # |
212779-48-1
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| PubChem CID |
2856
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
587.7±60.0 °C at 760 mmHg
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| Flash Point |
309.2±32.9 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.688
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| LogP |
1.54
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
24
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| Complexity |
401
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=CC(NC2=NC(NCCO)=NC3=C2N=CN3C(C)C)=CC=C1
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| InChi Key |
XZEFMZCNXDQXOZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H19ClN6O/c1-10(2)23-9-19-13-14(20-12-5-3-4-11(17)8-12)21-16(18-6-7-24)22-15(13)23/h3-5,8-10,24H,6-7H2,1-2H3,(H2,18,20,21,22)
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| Chemical Name |
2-[[6-(3-chloroanilino)-9-propan-2-ylpurin-2-yl]amino]ethanol
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| Synonyms |
NG 52; NG52; NG-52; Compound 52
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 69~75 mg/mL (199~216.3 mM)
Ethanol: ~23 mg/mL (~66.3 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8834 mL | 14.4171 mL | 28.8342 mL | |
| 5 mM | 0.5767 mL | 2.8834 mL | 5.7668 mL | |
| 10 mM | 0.2883 mL | 1.4417 mL | 2.8834 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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